Natural killer (NK) cells are best known for their capacity to kill tumor targets but they are clearly important in innate responses to infection, bone marrow transplant rejection, and autoimmune phenomena. In order to dissect their roles in immune responses, it will be important to determine the molecular basis for their target cell recognition. A major advance in understanding NK cell recognition came from the observation that NK cells are inhibited by MHC class I expression on targets leading Krre to propose the "missing-self hypothesis" in which NK cells are prevented from killing by MHC class I. The missing-self hypothesis appears to be explained by studies from the applicant's laboratory and others indicating that NK cells possess MHC class I-specific inhibitory receptors that block NK cell stimulation through a second type of receptor responsible for activation. However, the hypothesis has not been rigorously tested, particularly since further advances from their laboratory and others suggest that NK cell recognition, specificity, and activation now appear to be dependent on the interplay between the function of inhibitory, activation, and potential newly described costimulatory receptors. Furthermore, much less is known about the activation receptors and putative costimulatory receptors. In the previous granting period, the applicant's laboratory made substantial progress in characterizing mouse NK cell activation receptors belonging to the Ly49 family. Whereas the first defined Ly49 receptor, Ly49A, is a prototypical NK cell inhibitory receptor, Ly49D is an activation receptor that is specifically responsible for killing of Chinese hamster ovary (CHO) cells. Preliminary data indicate that they have identified and cloned a hamster ligand for Ly49D. Furthermore, functional studies suggest that the mouse H2Dd molecule also appears to be a ligand for Ly49D but there is no evidence for physical interaction between Ly49D and H2Dd, requiring further explanation. The applicant therefore proposes three specific aims: 1) Analyze ligand specificity of the Ly49D activation receptor; 2) Evaluate regulation of Ly49D activation by costimulation; and 3) Examine the "missing-self" hypothesis in vitro and in vivo. These studies will provide significant new insight into the function and specificity of NK cells.